Antigen fragments for the diagnosis of Toxoplasma gondii

ABSTRACT

The invention described herein relates to a method for identifying antigen fragments of  Toxoplasma gondii  proteins, and their use as diagnostic and immunogenic agents. Said method is implemented by means of selection of DNA fragments libraries of the parasite with sera of subjects who have been infected, using the phage display technique, and is characterised in that it uses the expression/exposure vector λKM4. The method allows also to identify antigen fragments related to the time of the infection.

This application is the US national phase of international applicationPCT/IT03/00162 filed 18 Mar. 2003, which designated the US and claimsbenefit of IT Application No. RM02A000159 filed 21 Mar. 2002 and ITApplication No. RM02A000568 filed 13 Nov. 2002, the entire contents ofeach of which are incorporated herein by reference.

The invention described herein relates to a method for identifyingantigen fragments of Toxoplasma gondii proteins, and their use asdiagnostic and immunogenic agents. Said method is implemented by meansof selection of cDNA libraries of the parasite or of DNA fragments ofspecific genes of the parasite with sera of subjects who have beeninfected by the parasite, using the phage display technique, and ischaracterised in that it uses the vector λKM4.

The invention described herein also relates to the technical field ofthe preparation of diagnostic means not applied directly to the animalor human body and furnishes compounds, methods for their preparation,methods for their use and compositions containing them which aresuitable for industrial application in the pharmaceutical and diagnosticfield, particularly for the detection and diagnosis of Toxoplasma gondiiinfections, as well as for the treatment and prevention of saidinfections.

BACKGROUND TO THE INVENTION

Early diagnosis is a priority and highly desirable objective in allfields of medicament, particularly because it allows an appreciableimprovement in the patient's life and a concomitant saving on the partof health care systems or on the part of the actual patients. In theparticular case of the invention described herein, early diagnosis isthat of potential or existing Toxoplasma gondii infection in pregnantwomen, with particular concern for the health of the foetus, and ininfected subjects, particularly those with impaired immunity.

Toxoplasma gondii is an obligate intracellular parasite that infects allmammalian cells, including those of human subjects (McCabe andRemington, N. Engl. J. Med. 1988, 318–313–5), and other animal genera,e.g. birds. The life cycle of the parasite is complex and one maydistinguish between three stages of infection: tachyzoite (asexual),bradyzoite (in tissue cysts, asexual) and sporozoite (in oocysts, sexualreproduction). Transmission typically occurs through ingestion ofundercooked meat harbouring tissue cysts or vegetables contaminated withoocysts shed by cats. Human infection is generally asymptomatic andself-limiting in immunocompetent hosts. In contrast, in subjects withimpaired immunity (particularly those affected by AIDS), toxoplasmosisis a severe opportunist infection, which may give rise to encephalitiswith very serious outcomes (Luft, B. J., Remington J. S., 1992, Clin.Infect. Dis. 15, 211–22). Moreover, contracting primary infection duringpregnancy may lead to miscarriages or to severe foetal disease inmammals.

For an extensive overview of the problem of toxoplasmosis the reader isreferred to the specialistic medical literature.

Diagnosis of T. gondii infection is established by isolating themicro-organism in the blood or body fluids, identifying the parasite intissues, detecting specific nucleotide sequences with PCR, or bydetecting specific anti-T. gondii immunoglobulins produced by the hostin response to the infection (Beaman et al., 1995 Principles andPractice of Infectious Diseases 4th Ed., Churchill Livingstone Inc., NewYork, 2455–75; Remington J S et al. 1995, Infectious Diseases of theFetus and Newborn Infant, W. B. Saunders, Philadelphia, Pa., 140–267).

One of the main problems in diagnosing T. gondii infections has to dowith pregnant women. To implement suitable therapies in good time andavoid possible damage to the foetus it is very important to establish ifparasitic infection occurred before or after conception. This isgenerally done by attempting to detect the presence of the variousclasses of anti-Toxoplasma immunoglobulins (IgG, IgM, IgA, avidity ofIgG). For this reason, the availability of specific, sensitivediagnostic agents is desirable.

T. gondii antigens have long been known and available, first of all asantigen mixtures obtained in various ways (FR 2,226,468, Mérieux; SU533376, Veterinary Research Institute; JP 54044016, Nihon ToketsuKanso), then as subsequent isolations of pure antigens (EP 0 082 745,Mérieux; EP 0 301 961, INSERM, Pasteur; WO 89/5658, Transgene) and theircharacterisation both as proteins, and of their respective genes (WO89/08700, U. Leland, Dartmouth Coll.; U.S. Pat. No. 4,877,726, Res.Inst. Palo Alto; WO 89/12683, INSERM, Pasteur; EP 0 391 319, MochidaPharm.; IT 1,196,817, CNR; EP 0 431 541, Behringwerke; WO 92/01067,CNRS; WO 92/02624, U. Flinders; WO 92/11366, Innogenetics, SmithklineBeecham; U.S. Pat. No. 5,215,917, Res. Inst. Palo Alto; WO 92/25689, FR2702491, INSERM, Pasteur; WO 96/02654, bioMeriéux, Transgene; EP 0 710724 Akzo; EP 0 724 016, bioMeriéux; EP 0 751 147, Behringwerke; U.S.Pat. No. 5,633,139, Res. Inst. Palo Alto; WO 97/27300, Innogenetics;U.S. Pat. No. 5,665,542, U.S. Pat. No. 5,686,575, Res. Inst. Palo Alto;WO 99/32633, Heska; JP 11225783, Yano; WO 99/61906, Abbott; WO 99/66043,Smithkline Beecham; JP 2000300278, Yano; WO 00/164,243, Virsol).

Numerous studies have found various different antigenic proteins of T.gondii and the gene sequences of these have also been determined.

Among the most interesting proteins both for diagnostic and therapeuticpurposes, in the form of vaccines, we should mention: the surfaceantigens SAG1 (or P30) (WO 89/08700, Stanford University; WO 89/12683Pasteur, INSERM; WO 94/17813, WO 96/02654, Transgene, bioMeriéux; EP 0724 016, WO 99/61906, U.S. Pat. No. 5,962,654, Harning et al., Clinicaland Diagnostic Laboratory Immunology, May 1996, 355–357); SAG2 (or P22)(Parmley et al., 1992, J. Clin. Microbiol. 30, 1127–33); the densegranule proteins GRA1 (or P24) (EP 0 301 961, Pasteur, INSERM; WO89/05658, Transgene, Cesbron-Delauw, et al., 1989 P.N.A.S. USA 86,7537–41); GRA2 (or P28) (WO 93/25689, INSERM, Pasteur; U.S. Pat. No.5,633,139, U.S. Pat. No. 5,665,542, U.S. Pat. No. 5,686,575, Res. Inst.Palo Alto; Prince et al., Mol. Biochem. Parasitol., 34 3–14); GRA4(Mevelec et al., Mol. Biochem. Parasitol. 56, 227–38); GRA6 (or P32) (FR2,702,491, INSERM, Pasteur; Lecordier al., Mol. Biochem. Parasitol. 70,85–94); GRA7 (WO 99/61906, Abbott; Jacobs et al., Mol. Biochem.Parasitol. 91, 237–49); GRA3 (Robben et al. 2002, J. Biol. Chem. 277,17544–47): the rhoptry antigens ROP1 (or P66) (U.S. Pat. No. 5,976,553,U. Leland; EP 0 431 541, Innogenetics); ROP2 (or P54) (Sharma et al., J.Immunol., 131, 377–83).

As described in the above-mentioned references, the antigens wereobtained with well-known recombinant cDNA techniques in expressionvectors. For example, for the antigen SAG1, WO 98/08700 uses knownexpression vectors in phage λgt11. WO 98/12683 uses the same phage andtransfects E. coli with a proprietary plasmid, or by preparing a specialexpression cassette, as in WO 96/02654. EP 0 724 016 obtains mimotypes,using combinatorial expression libraries of peptides. EP 0 301 961describes how to obtain excretion-secretion antigens with molecularweights ranging from 20 kDa to 185 kDa. WO 89/05658 describes a proteincontaining the epitopes of the 24 kDa protein recognised by theantibodies produced against Toxoplasma excretion-secretion antigens;this protein is obtained by transfection of cells by means of expressionvectors. The antigen P28 (GRA2) is described in U.S. Pat. No. 5,633,139and the method of obtaining it is again implemented through expressionin phage λgt11. The antigen P32 (GRA6) is described in patent FR2,702,491, the antigen ROP1 (P66) in U.S. Pat. No. 5,976,553, P35 (orGRA8) in EP 0 431 541, WO 99/57295 and WO 99/61906, and lastly P68 in EP0 431 541.

It should be stressed that all these antigens are obtained by means ofmolecular biology techniques that use the expression of proteins inbacterial cells. None of the documents cited describe the technique ofexpression/exposure of libraries of cDNA deriving from Toxoplasma gondiiin the lambda phage (phage display) to obtain fragments of antigens ofthe pathogen.

The invention described herein uses a new vector of DNA expression andprotein exposure as molecular fusion with the amino-terminal part ofprotein D of the lambda bacteriophage capsid (pD) (λKM4).

The expression/exposure vector was described for the first time inpatent application PCT/IT01/00405, filed on 26 Jul. 2001, the mostimportant part of which is incorporated herein. This vector, calledλKM4, differs from that used in expression only experiments (λgt11) inthat the recombinant protein coded for by the DNA fragment is expressedas fusion with a protein of the bacteriophage itself and then exposed onthe capsid. According to the vector project, the phage exposes theprotein fragment on the surface only if its open reading frame (ORF)coincides with pD. The size of the fragments of DNA cloned in ourlibraries was selected in order to represent a population of medium sizeranging from 300 to 1000 nucleotide base pairs (bp), and, forstatistical reasons, most of the out-of-frame sequences contain stopcodons which do not permit their translation and consequently exposureon the surface of the phage.

SUMMARY OF THE INVENTION

It has now been found that the combination of the affinity selection andphage display techniques, together with the use of the vector KM4,provides a method for the identification of specific antigen fragmentsof Toxoplasma gondii by means of the selection of display libraries ofDNA fragments with sera of infected individuals. DNA fragments areobtained either from cDNA of whole parasite or from DNA encoding forknown specific gene products. With this method it proves possible toidentify antigen fragments from very large libraries (i.e. expressing alarge number of different sequences). The antigen fragments thusidentified enable specific ligands to be obtained, which in turn can beused as diagnostic and therapeutic means.

Therefore, one object of the invention described herein is a method forthe identification of antigen fragments of Toxoplasma gondii proteins,by means of the selection of libraries of DNA fragments with sera ofsubjects who have been infected by the parasite, using the phage displaytechnique, characterised in that it uses the vector λKM4.

Another object of the present invention are antigen fragments obtainablewith the above-mentioned method, both isolated and characterised, and assets of antigen fragments called “collections”. The invention describedherein also extends to the antigen portions of said fragments(epitopes).

The use of said antigen fragments as diagnostic agents and the relateddiagnostic aids containing them, for example in the form of kits orother supports, constitute a further object of the present invention.

The use of said antigen fragments as active agents, particularly with animmunogenic action, for the preparation of medicaments for theprevention and therapy of Toxoplasma gondii infection, constitute afurther object of the present invention.

Another object of the present invention are the gene sequences codingfor the above-mentioned antigen fragments, their use as medicaments,particularly for the prevention and therapy of Toxoplasma gondiiinfection, e.g. as gene therapy. The present invention also extends tothe gene sequences that hybridise with the sequences of theabove-mentioned fragments in stringent hybridisation conditions.

Another object of the present invention are anti-epitope antibodies andtheir use in the preparation of diagnostic, preventive and therapeuticmeans, e.g. as conjugates with active ingredients such as chemo-therapyagents. Antibodies can be generated also against collections of saidepitopes.

The method provided by the present invention makes it possible toconfirm the use of the Toxoplasma gondii antigens described above assuch as diagnostic agents and also to identify in known antigens theepitopes that trigger an immune response in human subjects; this portionis a further object of the present invention; but it also makes itpossible to identify the antigenic function of proteins of Toxoplasmagondii, or of portions thereof, which, though their structure andpossibly their physiological function may be known, are unknown asregards their antigenic function, and such function comes within theframework of the present invention; lastly, the method according to thepresent invention also provides new antigen fragments of Toxoplasmagondii proteins, that constitute yet another object of the presentinvention.

Another object of the present invention is the use of the antigenfragments thus identified for the preparation of means of diagnosing theinfection, as well as the actual diagnostic means containing them. Theuse realtes also to the diagnosis of the time of the infection, inparticular by the IgG avidity assay.

Another object of the present invention is the use of the antigenfragments thus identified as medicaments, particularly for thepreparation of formulations, and particularly in the form of vaccines,which are useful for the prevention and cure of the infection. Thevaccines according to the present invention are suitable for use inhumans and other animals (particularly pigs, cats, sheeps).

Another object of the present invention are ligands generated from theabove-mentioned antigen fragments and the related collections and theuse of such ligands for the preparation of diagnostic means for thedetection of the infection, with particular reference to the time ofinfection, as well as therapeutic means for the prevention and treatmentof the infection itself.

Another object of the present invention is a method for the diagnosis ofToxoplasma gondii infection, comprising the selection of sera ofsubjects affected or suspected of being affected by said infection withthe above mentioned antigen fragments and/or their collection and/or atleast one ligand and/or antibody.

These and other objects will be illustrated here below in detail, alsoby means of examples and figures, where FIG. 1 represents the map of thevector λKM4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises the construction of expression/exposurelibraries of DNA fragments prepared from Toxoplasma gondii cells, theselection of such libraries with the sera of patients who have beeninfected by Toxoplasma gondii, the characterisation of the antigenfragments, and the use of said fragments for developing selectivediagnostic means.

Optionally, the present invention may entail the generation of specificligands for said antigen fragments (e.g. human recombinant antibodies orhumanised murine recombinant antibodies) and the construction ofselective diagnostic means that incorporate the ligands generated.

Antibodies and ligands of the present invention can be obtainedaccording according to the general common knowledge and conventionalmethods.

The method according to the present invention advantageously combinesaffinity selection and the power of phage display.

What is meant by phage display, as understood by the person of ordinaryskill in the art, is a strategy based on the selection ofexpression/exposure libraries in which small protein domains are exposedon the surface of bacteriophages containing the corresponding geneticinformation.

The method implemented according to the present invention for the firsttime provides new and advantageous analysis possibilities:

-   -   the use of small amounts of serum to identify antigen fragments        of the infectious agent,    -   the possibility of selecting only the domains responsible for        the interaction with the antibodies, without having to express        the entire gene, the product of which may be insoluble or toxic;

lastly, the possibility of effecting successive cycles of selectionusing sera from different patients or mixtures of sera facilitates theidentification of cross-reactive antigens which represent one of themain objectives of the present invention.

For the above-mentioned reasons, for each library, messenger RNA waspurified from an adequate number of cells (e.g. 10⁶ cells), using commoncommercially available means, from which the corresponding cDNA wasgenerated. The latter was fragmented (by means of the bacterial enzymeDNaseI) and then cloned in the expression/exposure vector λKM4 (seeexample).

In the other embodiment, relating to specific T. gondii gene, eachspecific T. gondii gene, was amplified from the DNA of the parasite(either cDNA or genomic DNA, both prepared by using common commerciallyavailable kits) by means of PCR with specific syntheticoligonucleotides. DNA of single genes was then fragmented randomly bymeans of the bacterial enzyme DNaseI and then cloned as a pool in theexpression/exposure vector λKM4.

The amplification of the libraries was done by means of normaltechniques with which the expert in the field is familiar, e.g. byplating, growth, elution, purification and concentration (Sambrook etal., 1989, Molecular Cloning: a laboratory manual, Cold Spring HarborLaboratory Press, NY). The libraries were then used to develop theselection conditions, screening and characterisation of the sequencesidentified. Lastly, the phage clones identified were characterised byimmunoenzymatic assays.

A library of the phage display type, constructed using cDNA derivingfrom cells of pathogenic organisms, makes it possible to exploitaffinity selection, which is based on incubation of specific sera(reactive with the pathogen) with collections of bacteriophages thatexpress portions of proteins of the pathogen on their capside and thatcontain the corresponding genetic information. The bacteriophages thatspecifically bind the antibodies present in the serum are easilyrecovered, remaining bound (by the antibodies themselves) to a solidsupport (e.g. magnetic beads); the non-specific ones, by contrast, arewashed away. Direct screening, i.e. the analysis of the ability ofsingle phage clones to bind the antibodies of a given serum, is doneonly at a later stage, when the complexity of the library (i.e. thedifferent number of sequences) is substantially reduced, precisely as aresult of the selection.

The use of selection strategies allows faster analysis of a large numberof different protein sequences for the purposes of identifying thosethat respond to a particular characteristic, e.g. interactingspecifically with antibodies present in the serum of patients who havebeen infected by the pathogen. What is more, the combination of affinityselection and phage display makes it possible to use a smaller amount ofserum for each analysis. The direct screening of a classic cDNA library,in fact, entails the use of large amounts of serum, which are not alwayseasy to obtain. For example, to analyse a library of approximately 10⁶independent clones it would be necessary to incubate along with thepreselected serum the numerous filters containing a total ofapproximately 10⁷ phage plaques transferred from the different cultureplates with the infected bacteria (e.g. a serum volume of 1–10 ml). Theuse of a display-type library, on the other hand, permits affinityselection in small volumes (0.1–1 ml) prior to direct screening, andfrom limited amounts of serum, such as, for example, 10 μl.

Lastly, given that a very large number of bacteriophages can becontained in small volumes (e.g. 10¹¹ phage particles are normallycontained in a volume of 0.1 ml), and affinity selection is done insmall volumes (0.1–1 ml), a further advantage of the use of display-typelibraries consists in analysing a number of independent clones(particles of recombinant phages exposing different cDNA sequences ontheir surfaces) 10–100 times greater (e.g. 10⁸ different bacteriohages)than expression-alone libraries where, as a result of technicalproblems, not more than 10⁶ independent clones are normally analysed.

As regards industrial applicability, one possible realisation of thepresent invention is in the form of diagnostic kits containing theantigen fragments and/or ligands and/or antibodies described above.

The diagnostic kits which are the object of the present invention areknown to the expert in the field and do not require any particulardescription. By way of an example, the reader is referred to the patentliterature cited above, to which may be added U.S. Pat. No. 6,265,176and WO 01/63283 as further references.

Similar considerations hold good for the therapeutic application, wherethe preparation of medicaments or vaccines comes within the framework ofgeneral knowledge; for further reference purposes the reader is againreferred to the patent literature cited in the present description.

The invention will now be illustrated in greater detail by means ofexamples and figures, where FIG. 1 presents the map of the vector λKM4.

EXAMPLE 1

Construction of the Vector λKM4

This technique is described in international patent application No.PCT/IT01/00405, filed on 26 Jul. 2001 and incorporated herein forreference purposes, explicitly mentioning the references cited therein.FIG. 1 represents the map of the vector λKM4. The plasmid pNS3785(Sternberg and Hoess, 1995, Proc. Natl. Acad. Sci. USA., 92:1609–1613)was amplified by inverse PCR using the synthetic oligonucleotides5′-TTTATCTAGACCCAGCCCTAGGAAGCTTCTCCTGAGTAGGACAAATCC-3′ (SEQ ID No 1)bearing the sites XbaI and AvrII (underlined) for the subsequent cloningof the lambda phage, and 5′-GGGTCTAGATAAAACGAAAGGCCCAGTCTTTC-3′ (SEQ IDNo 2) bearing the site XbaI. In inverse PCR a mixture of Taq DNApolymerase and Pfu DNA polymerase was used to increase the fidelity ofthe DNA synthesis. Twenty-five amplification cycles were performed (95°C.-30 sec, 55° C.-30 sec, 72° C.-20 min). The autoligation of the PCRproduct, previously digested with XbaI endonuclease gave rise to theplasmid PKM3. The lambda gene pD was amplified with PCR from the plasmidpNS3785 using the primers5′-CCGCCTTCCATGGGTACTAGTTTTAAATGCGGCCGCACGAGCAAAGAAACCTTTAC-3′ (SEQ IDNo 3) e 5′-AGCTTCCTAGGGCTGGGTCTAG-3′ (SEQ ID No 4) containing therestriction sites NcoI, SpeI, NotI and EcoRI, respectively,(underlined). The PCR product was then purified, digested with NcoI andEcoRI endonuclease and recloned in sites NcoI and EcoRI of pKM3,resulting in the plasmid pKM4 bearing only the restriction sites SpeIand NotI at the 5′ end of the protein gpD. The plasmid was then digestedwith XbaI endonuclease and cloned in the XbaI site of the lambda phageDam15imm21nin5 (Sternberg and Hoess, 1995, Proc. Natl. Acad. Sci. USA.,92:1609–1613).

Construction of cDNA Library from Tachyzoites of Toxoplasma gondii

Tachyzoites of the protozoon Toxoplasma gondii (RH strain) were grown invitro in monkey kidney cells (“VERO” African green monkey cells) usingDMEM culture medium containing 10% foetal bovine serum, 2 mM glutamineand 0.05 mg/ml gentamicin (Gibco BRL, Canada). The parasites werecollected after complete lysis of the host cells and purified byfiltration (filter porosity 3 μm) followed by centrifuging. 4 μg of mRNAwere isolated from 10⁷ tachyzoites using the “QuickPrep Micro mRNAPurification Kit” (Amersham Pharmacia Biotech, Sweden) and following themanufacturer's instructions. The double-helix cDNA was synthesised from200 ng of poly(A)⁺ RNA using the “SMART cDNA Library Construction Kit”(Clontech, CA, USA) and following the manufacturer's instructions. 10 μgof total cDNA were then fragmented randomly using 0.5 ng of theendonuclease DNaseI (Sigma-Aldrich, USA). The mixture of cDNA and DNaseIwas incubated for 20 minutes at 15° C. and the cDNA fragments werepurified with extraction in phenol/chloroform and subsequentpurification by means of the “QIAquick PCR Purification Kit” (Qiagen,CA, USA), following the manufacturer's instructions. The 3 μg ends ofthe cDNA fragments were “flattened” by incubating the DNA with 9 unitsof the enzyme T4 DNA polymerase (New England Biolabs, MA, USA) for 60minutes at 15° C. The fragments were then purified by means ofextraction in phenol/chloroform and subsequent precipitation in ethanol.500 ng of the resulting DNA were bound with a 20-fold molar excess of“synthetic adaptors” for the purposes of adding the restriction sitesSpeI and NotI to the ends of the fragments. Six adaptors were used,obtained by hybridisation of the following pairs of oligonucleotides:K185 5′-CTAGTCGTGCTGGCCAGC-3′ (SEQ ID No 5) and K1865′-GCTGGCCAGCACGA-3′ (SEQ ID No 6); K187 5′-CTAGTCGTGCTGGCCAGCT-3′(SEQID No 7) and K188 5′-AGCTGGCCAGCACGA-3′ (SEQ ID No 8); K1895′-CTAGTCGTGCTGGCCAGCTG-3′ (SEQ ID No 9) and K190 5′-CAGCTGGCCAGCACGA-3′(SEQ ID No 10); K191 5′-TCTGGTGGCGGTAGC-3′ (SEQ ID No 11) and K1925′-GGCCGCTACCGCCACCAGA-3′ (SEQ ID No 12); K1935′-TTCTGGTGGCGGTAGC-3′(SEQ ID No 13) and K194 5′-GGCCGCTACCGCCACCAGAA-3′(SEQ ID No 14); K195 5′-TTTCTGGTGGCGGTAGC-3′ (SEQ ID No 15) and K1965′-GGCCGCTACCGCCACCAGAAA-3′(SEQ ID No 16). The excess of unligatedadaptors was removed from the ligation mixture by electrophoresis on 2%agarose gel and the cDNA fragments with molecular weights ranging from300 bp to 1000 bp were excised from the gel and purified by means of the“Qiaquick gel extraction kit” (Qiagen, Calif., USA) following themanufacturer's instructions. The vector λKM4 was digested with SpeI/NotIand for the construction of the library 6 ligation mixtures wereperformed, each containing 0.4 μg of vector and approximately 7 ng ofinsert. After overnight incubation at 4° C. the ligation mixtures werepackaged in vitro with the “Ready-To-Go lambda packaging kit” (AmershamPharmacia Biotech, Sweden) and plated for infection of BB4 cells(bacterial cells of E. coli strain BB4; Sambrook et al., 1989, MolecularCloning: a laboratory manual, Cold Spring Harbor Laboratory Press, NY).After overnight incubation at 37° C. the phage was eluted from theplates with SM buffer (Sambrook et al., 1989, Molecular Cloning: alaboratory manual, Cold Spring Harbor Laboratory Press, NY), purified,concentrated and stored at −80° C. in SM buffer containing 7%dimethylsulphoxide. The complexity of the library calculated as thenumber of total independent clones with inserts was 10⁷ clones.

Affinity Selection

Two distinct methods were used for selecting the phage library withhuman sera. In the first method 100 μl of magnetic beads coated withProtein G (Dynabeads Protein-G, Dynal, Norway) were incubated with 10 μlof human serum for 30 minutes at room temperature. The beads were thenincubated for 1 hour at 37° C. with blocking solution consisting of: 5%skimmed milk powder in PBS (Sambrook et al., 1989, Molecular Cloning: alaboratory manual, Cold Spring Harbor Laboratory Press, NY), 0.05% Tween20, and MgSO₄ 10 mM. Approximately 10¹⁰ phage particles of the librarywere added to the beads and diluted in 1 ml of blocking solution for afurther 4-hour incubation at room temperature with weak stirring. In thesecond method 40 μl of “M280-Tosyl activated” magnetic beads (Dynal,Norway) were coated with human anti-IgM antibodies (Sigma-Aldrich, USA)following the manufacturer's instructions. The beads were then washedwith PBS/TritonX-100 1% and incubated with 10 μl of human serum in 300μl of blocking solution for 2 hours at room temperature. After washingthe beads three times with washing solution (PBS, 1% TritonX100, 10 mMMgSO₄), 10¹⁰ phage particles of the library were added to the beads anddiluted in 200 μl of blocking solution for a further 3-hour incubationat room temperature with weak stirring.

With both selection methods, the beads were washed 10 times with 1 ml ofwashing solution (PBS, 1% TritonX100, 10 mM MgSO₄). The boundbacteriophages were amplified for infection of BB4 cells added directlyto the beads (1.2 ml per selection) and subsequent 30-minute incubationat room temperature. 12 ml of NZY-Top Agar (Sambrook et al., 1989,Molecular Cloning: a laboratory manual, Cold Spring Harbor LaboratoryPress, NY) were added to the mixture of beads and cells and immediatelypoured onto NZY plates (2 15-cm Petri capsules for selection). Theplates were incubated for 12–16 hours at 37° C. The next day the phageswere collected from the plates by means of the addition of 15 ml of SMbuffer per plate and stirring for 4 hours at room temperature. Thephages were purified by precipitation in PEG/NaCl (20% polyethyleneglycol, NaCl 1M) and finally resuspended in 5 ml of SM and stored at +4°C.

Selection of the Library with Human Sera

To identify the specific antigens of T. gondii an affinity selectionprocedure was used consisting of two “panning” cycles with one or morepositive sera (that is to say sera deriving from a patient who testedpositive for the presence of antibodies directed against the parasite),followed by an immunological screening procedure carried out with thesame sera or, alternatively, by analysis of single clones taken atrandom from the mixture of selected phages. Preferably, the library wasselected with 10 positive sera (T1, T2, T3, T4, T5, T6, T7, T8, T9 andT10), generating, after a single selection cycle, the correspondingmixtures p1^(I), p2^(I), p3^(I), p4^(I), p5^(I), p6^(I), p7^(I), p8^(I),p9^(I) and p10^(I). Each mixture was then subjected to a second affinityselection cycle with the same serum, according to the first strategymentioned above, giving rise to a second series of mixtures (calledp1^(II), p2^(II), p3^(II), p4^(II), p5^(II), p6^(II), p7^(II), p8^(II),p9^(II) and p10^(II)). The initial characterisation by means of anenzyme-linked immunosorbent assay (Phage-ELISA) showed that some of themixtures were more reactive with the corresponding serum used for theselection, thus confirming the efficacy of the library and the affinityselection procedure. Various positive clones were identified by means ofimmunoplate screening per plaque of reactive mixtures.

Phage-ELISA

Multi-well plates (Immunoplate Maxisorb, Nunc, Denmark) were coated,incubating 100 μl/well of anti-lambda polyclonal antibodies overnight at4° C. with a concentration of 0.7 μg/ml in NaHCO₃ 50 mM, pH 9.6. Aftereliminating the coating solution, the plates were incubated with 250 μlof blocking solution (5% skimmed milk powder in PBS, 0.05% Tween-20).The plates were then washed twice with washing buffer (PBS, 0.05%Tween-20). A mixture of 100 μl of blocking solution containing phagelysate (diluted 1:1) was added to each well and incubated for 60 minutesat 37° C. 1 μl of human serum was preincubated for 30 minutes at roomtemperature with 10⁹ wild-type phage particles, 1 μl of rabbit serum, 1μl of bacterial extract of BB4 cells, 1 μl of foetal bovine serum in 100μl of blocking solution. The plates were washed 5 times after incubationwith the phage lysate and then incubated with the serum solution for 60minutes at 37° C. The plates were then washed 5 times and incubated inblocking solution containing human anti-immunoglobulin antibodiesconjugated with the enzyme peroxidase (Sigma-Aldrich, USA) diluted1:10000 and rabbit serum diluted 1:40. After 30 minutes' incubation theplates were washed 5 times and the peroxidase activity was measured with100 μl of TMB liquid substrate (Sigma-Aldrich, USA). After 15 minutes'development, the reaction was stopped by adding 25 μl of H₂SO₄ 2M.Lastly, the plates were analysed using an automatic ELISA reader(Multiskan, Labsystem, Finland) and the results were expressed asOD=OD_(450 nm)−OD_(620 nm). The ELISA data were assessed as mean valuesof two independent assays.

Immunoscreening

Phage plaques were transferred from the bacterial medium tonitrocellulose filters (Schleicher & Schuell, Germany) by means ofincubation at room temperature for 60 minutes. The filters were blockedfor 60 minutes at room temperature in blocking solution (5% skimmed milkpowder in PBS, 0.05% Tween-20). 40 μl of human serum were preincubatedwith 40 μl of bacterial extract of BB4 cells, 10⁹ wild-type lambda phageparticles in 4 ml of blocking solution. After eliminating the blockingsolution, the filters were incubated with the serum for 3 hours at roomtemperature under stirring. The filters were then washed 5 times withwashing buffer (PBS, 0.05% Tween-20) and then incubated for 60 minutesat room temperature, alternatively with human anti-IgG antibodiesconjugated with alkaline phosphatase (Sigma-Aldrich, USA), or with humananti-IgM antibodies conjugated with alkaline phosphatase (Sigma-Aldrich,USA), both diluted 1:7500 in blocking solution. After washing thefilters 5 times, 5 ml of development solution (substrates BCIP and NBT,Sigma-Aldrich, USA) were added and the development was interrupted bywashing the filters in water.

Preparation of the Lambda Phage from Lysogenic Cells

Phage clones that proved positive at immunoscreening (direct screening)were isolated from the respective phage plaques and then amplified forsubsequent characterisation. The bacterial BB4 cells were grown understirring at 37° C. up to an optical density OD₆₀₀=1.0 in LB culturemedium (Sambrook et al., 1989, Molecular Cloning: a laboratory manual,Cold Spring Harbor Laboratory Press, N.Y.) containing 0.2% maltose and10 mM MgSO₄, recuperated by centrifuging and resuspended in SM buffer atoptical density OD₆₀₀=0.2. 100 μl of cells were infected withrecombinant bacteriophages recovered from single plaques, incubated for20 minutes at room temperature, plated on LB medium with ampicillin (100μg/ml) and then incubated for 18–20 hours at 32° C. A single bacterialcolony was then grown in 10 ml of LB/ampicillin overnight at 32° C.under stirring. 500 ml of LB/ampicillin and MgSO₄ 10 mM were added to 5ml of the overnight culture and incubated at 32° C. up to an opticaldensity OD₆₀₀=0.6 under vigorous stirring. The culture was thenincubated for 15 minutes in a water bath at 45° C. and then at 37° C.for a further 3 hours. After this, the bacteriophages were purified ofthe bacterial culture according to standard procedures (Sam brook etal., 1989, Molecular Cloning: a laboratory manual, Cold Spring HarborLaboratory Press, NY) and stored at +4° C.

Lastly, the phage clones were analysed by means of phage-ELISA with asubstantial panel of positive and negative sera. Clones whose ELISAvalue exceeded the background value, as obtained from the sum of themean of the measurements of the negative sera and three times thestandard deviation, were judged to be positive.

The following table 1 gives, by way of examples, the reactivity of anumber of the recombinant bacteriophages selected.

TABLE 1 Reactivity of phage Reactivity of phage clone clone withpositive sera with negative sera Name of clone (positive/total pos.)(negative/total neg.) Tx-4.11 13/21 0/10 TxM-17.2 4/8 1/8  Tx-15.1111/21 0/10 Tx-1.11 19/21 0/10 Tx-8.0  6/21 0/10 Tx-1.16 20/21 0/10Tx-9.18  9/21 0/8  Tx-7.11 21/21 0/10Characterisation of Positive Clones

The clones which showed multiple reactivity with the Toxoplasma gondiipositive sera and which presented no reactivity to the negative serawere subsequently sequenced and compared with various databases ofsequences currently available (Non-Redundant Genbank CDS, Non-RedundantDatabase of Genbank Est Division, Non-Redundant Genbank+EMBL+DDBJ+PDBSequences).

The sequences obtained can be classified in four groups:

-   -   sequences that code for known T. gondii antigen fragments;    -   sequences that code for known proteins which, however, are not        known to be involved in the human antibody response;    -   sequences that code for unknown proteins (e.g. EST);    -   new sequences, not yet figuring in the databases.

The following table 2 gives, by way of examples, the sequences of someof the clones selected:

TABLE 2 Name Identi- Classi- of clone Sequence fication fication Tx-4.11AGTGGAGGGACAGGGCAGGGATTA GRA 1 Dense (SEQ ID GGAATCGGAGAATCTGTAGATTTGknown granule No 17) GAGATGATGGGGAACACGTATCGT T. gondii proteinGTGGAGAGACCCACAGGCAACCCG antigen GACTTGCTCAAGATCGCCATTAAAGCTTCAGATGGATCGTACAGCGAA GTCGGCAATGTTAACGTGGAGGAGGTGATTGATACTATGAAAAGCATG CAGAGGGACGAGGACATTTTCCTTCGTGCGTTGAACAAAGGCGAAACA GTAGAGGAAGCGATCGAAGACGTGGCTCAAGCAGAAGGGCTTAATTCG GAGCAAACCCTGCAACTGGAAGATGCAGTGAGCGCGGTGGCGTCTGTT GTTCAAGACGAG TxM-17.2 TACTCTTCACCACGAATAGTTGTTGRA2 Dense (SEQ ID TTGATTAGATATTGCTTCTTCTCC known granule No 18)ACATATCGCCTCACAATGTTCGCC T. gondii protein GTAAAACATTGTTTGCTGGTTGTTantigen GCCGTTGGCGCCCTGGTCAACGTC TCGGTGAGGGCTGCCGAGTTTTCCGGAGTTGTTAACCAGGGACCT Tx-15.11 GCTGCCTTGGGAGGCCTTGCGGCG GRA 3 Dense (SEQID GATCAGCCTGAAAATCATCAGGCT granule No 19) CTTGCAGAACCAGTTACGGGTGTGprotein— GGGGAAGCAGGAGTGTCCCCCGTC unknown AACGAAGCTGGTGAGTCATACAGT asanti- TCTGCAACTTCGGGTGTCCAAGAA gen in GCTACCGCCCCAGGTGCAGTGCTC humanCTGGACGCAATCGATGCCGAGTCG response GATAAGGTGGACAATCAGGCGGAGGGAGGTGAGCGTATGAAGAAGGTC GAAGAGGAGTTGTCGTTATTGAGGCGGGAATTATATGATCGCACAGAT CGCCCTGGT Tx-1.11 CAGTTCGCTACCGCGGCCACCGCG GRA7 Dense (SEQ ID TCAGATGACGAACTGATGAGTCGA known granule No 20)ATCCGAAATTCTGACTTTTTCGAT T. gondii protein GGTCAAGCACCCGTTGACAGTCTCantigen AGACCGACGAACGCCGGTGTCGAC TCGAAAGGGACCGACGATCACCTCACCACCAGCATGGATAAGGCATCT GTAGAGAGTCAGCTTCCGAGAAGAGAGCCATTGGAGACGGAGCCAGAT GAACAAGAAGAAGTTCAT Tx-8.0GAGAACCCGGTGAGACCGCCTCCT GRA8 Dense (SEQ ID CCCGGTTTCCATCCAAGCGTTATTknown granule No 21) CCCAATCCCCCGTACCCGCTGGGC T. gondii proteinACTCCAGCGGGCATGCCACAGCCA antigen GAGGTTCC Tx-1.16AGGAGGACTGGATGTCATGCCTTC MIC 3 Micro- (SEQ ID AGGGAGAACTGCAGCCCTGGTAGAneme No 22) TGTATTGATGACGCCTCGCATGAG protein— AATGGCTACACCTGCGAGTGCCCCunknown ACAGGGTACTCACGTGAGGTGACT as anti- TCCAAGGCGGAGGAGTCGTGTGTG genin GAAGGAGTCGAAGTCACGCTGGCT human GAGAAATGCGAGAAGGAATTCGGC responseATCAGCGCGTCATCCTGCAAATGC GAT Tx-9.18 GCACCCACTCAATCTGAAATGAAA MIC 5Micro- (SEQ ID GAATTCCAAGAGGAAATCAAAGAA known neme No 23)GGGGTGGAGGAAACAAAGCATGAA T. gondii protein GACGATCCTGAGATGACGCGGCTCantigen ATGGTGACCGAGAAGCAGGAGAGC AAAAATTTCAGCAAGATGGCGAAATCCCAGAGTTTTAGCACGCGAATC GAAGAGCTCGGGGGATCCATTTCGTTTCTAACTGAAACGGGGGTCACA ATGATCGAGTTGCCCAAAACTGTCAGTGAACATGACATGGACCAACTA CTCCAC Tx-7.11 GTTATGGCATCGGATCCCCCTCTT SAG 1Surface (SEQ ID GTTGCCAATCAAGTTGTCACCTGC known protein No 24)CCAGATAAAAAATCGACAGCCGCG T. gondii GTCATTCTCACACCGACGGAGAAC antigenCACTTCACTCTCAAGTGCCCTAAA ACAGCGCTCACAGAGCCTCCCACTCTTGCGTACTCACCCAACAGGCAA ATCTGCCCAGCGGGTACTACAAGTAGCTGTACATCAAAGCTGTAACAT TGAGCTCCTTGATTCCTGAAGCAGAAGATAGCTGGTGGACGGGGGATT CTGCTAGTCTCGACACGGCAGGCATCAAACTCACAGTTCCAATCGAGA AGTTCCCCGTGACAACGCAGACGTTTGTGGTCGGTTGCATCAAGGGAG ACGACGCACAGAGTTGTATGGTCACGGTGACAGTACAAGCCAGAGCCT CATCGGTCGTCAATAATGTCGCAAGGTGCTCCTATGGTGCGGACAGC Tx-4.18 CCATCGGTCGTCAATAATGTCGCA SAG 1 Surface(SEQ ID AGGTGCTCCTACGGTGCAGACAGC known protein No 25)ACTCTTGGTCCTGTCAAGTTGTCT T. gondii GCGGAAGGACCCACTACAATGACC antigenCTCGTGTGCGGGAAAGATGGAGTC AAAGTTCCTCAAGACAACAATCAGTACTGTTCCGGGACGACGCTGACT GGTTGCAACGAGAAATCGTTCAAAGATATTTTGCCAAAATTAACTGAG AACCCGTGGCAGGGTAACGCTTCGAGTGATAAGGGTGCCACGCTAACG ATCAAGAAGGAAGCATTTCCAGCCGAGTCAAAAAGCGTCATTATTGGA TGCACAGGGGGATCGCCTGAGAAGCATCACTGTACCGTGAAACTGGAG TTTGCCGGGGCTGCAGGGTCAGCA AAATCGGCT

The clone Tx-4.11 constitutes a fragment of the antigen GRA1(Cesbron-Delauw et al., 1989, Proc. Natl. Acad. Sci. USA 86:7537–7541)but has never been identified as an “antigen fragment” of the protein inthe human humoral response. Said clone has the amino acid sequence (SEQID No 26)SGGTGQGLGIGESVDLEMMGNTYRVERPTGNPDLLKIMKASDGSYSEVGNVNVEEVIDTMKSMQRDEDIFLRALNKGETVEEAIEDVAQAEGLNSEQTLQLEDAVSAVASVVQDE and its use as a fragmentcontaining an epitope is covered by the present invention.

The clone TxM-17.2 constitutes a fragment of the antigen GRA2 (Prince etal., 1989, Mol. Biochem. Parasitol., 34: 3–14) but has never beenidentified as an “antigen fragment” of the protein in the human humoralresponse. Said clone has the amino acid sequence (SEQ ID No 27)YSSPRIVVLIRYCFFSTYRLTMFAVKHCLLVVAVGALVNVSVRAAEFSGVVNQGP and its use afragment containing an epitope is covered by the present invention.

The clone Tx-15.11 constitutes a fragment of the gene GRA3 (Bermudes etal., 1994, Mol. Biochem. Parasitol., 68: 247–257) and has never beenidentified as an antigen in the human antibody response. Said clone hasthe amino acid sequence (SEQ ID No 28)AALGGLAADQPENHQALAEPVTGVGEAGVSPVNEAGESYSSATSGVQEATAPGAVLLDAIDAESDKVDNQAEGGERMKKVEEELSLLRRE LYDRTDRPG and its use as afragment containing an epitope is covered by the present invention.

The clone Tx-1.11 constitutes a fragment of the antigen GRA7 (Bonhommeet al., 1998, J. Histochem. Cytochem. 46, 1411–1421) and has never beenidentified as an “antigen fragment” of the protein in the human humoralresponse. Said clone has the amino acid sequence (SEQ ID No 29)FATAATASDDELMSRIRNSDFFDGQAPVDSLRPTNAGVDSKGTDDHLTTSMDKASVESQLPRREPLETEPDEQEEVHFand its use as a fragment containing an epitope is covered by thepresent invention.

The clone Tx-8.0 constitutes a fragment of the antigen GRA8 (Kimberly etal., 2000, Mol. Biochem. Parasitol., 105: 25–37) and has never beenidentified as an “antigen fragment” of the protein in the human humoralresponse. Said clone has the amino acid sequence (SEQ ID No 30)ENPVRPPPPGFHPSVIPNPPYPLGTPAGMPQPEVP and its use as a fragment containingan epitope is covered by the present invention.

The clone Tx-1.16 constitutes a fragment of the gene MIC3 (Garcia-Réguetet al., 2000, Cellular Microbiol., 2: 353–364) and has never beenidentified as an antigen in the human antibody response. Said clone hasthe amino acid sequence (SEQ ID No 31)RRTGCHAFRENCSPGRCIDDASHENGYTCECPTGYSREVTSKAEESCVEGVEVTLAEKCEKEFGISASSCKCD and its use as a fragment containing an epitope is coveredby the present invention.

The clone Tx-9.18 constitutes a fragment of the antigen MIC5 (Brydges etal., 2000, Mol. Biochem. Parasitol., 111: 51–66) but has never beenidentified as an “antigen fragment” of the protein in the human humoralresponse. Said clone has the amino acid sequence (SEQ ID No 32)APTQSEMKEFQEEIKEGVEETKHEDDPEMTRLMVTEKQESKNFSKMAKSQSFSTRIEELGGSISFLTETGVTMIELPKTVSEHDMDQLLH and its use as a fragment containing an epitope is covered by thepresent invention.

The clone Tx-7.11 constitutes a fragment of the antigen SAG1 (Burg etal., 1988, J. Immunol., 141:3584–3591) but has never been identified asan “antigen fragment” of the protein in the human humoral response. Saidclone has the amino acid sequence (SEQ ID No 33)VMASDPPLVANQVVTCPDKKSTAAVILTPTENHFTLKCPKTALTEPPTLAYSPNRQICPAGTTSSCTSKAVTLSSLIPEAEDSWWTGDSASLDTAGIKLTVPIEKFPVTTQTFVVGCIKGDDAQSCMVTVTVQARASSVVNNVARCSYG ADS and its use afragment containing an epitope is covered by the present invention.

The clone Tx-4.18 constitutes a fragment of the antigen SAG1 (Burg etal., 1988, J. Immunol., 141:3584–3591) but has never been identified asan “antigen fragment” of the protein in the human humoral response. Saidclone has the amino acid sequence (SEQ ID No 34)PSVVNNVARCSYGADSTLGPVKLSAEGPTTMTLVCGKDGVKVPQDNNQYCSGTTLTGCNEKSFKDILPKLTENPWQGNASSDKGATLTIKKEAFPAESKSVIIGCTGGSPEKHHCTVKLEFAGAAGSAKSA and its use as a fragment containing anepitope is covered by the present invention.

Expression of cDNA Fragments Selected from the Library as FusionProducts with GST

The plasmid pGEX-SN was constructed by cloning the DNA fragment derivingfrom the hybridisation of the synthetic oligo-nucleotides K1085′-GATCCTTACTAGTTTTAGTAGCGGCCGCGGG-3′ (SEQ ID No 35) and K1095′-AATTCCCGCGGCCGCTACTAAAACTAGTAAG-3′ (SEQ ID No 36) in the BamHI andEcoRI sites of plasmid pGEX-3X (Smith and Johnson, 1988, Gene, 67,31–40).

The phage clones for which specific reactivity with sera of patientstesting positive for Toxoplasma gondii was demonstrated, were amplifiedand then analysed with a substantial panel of positive and negativesera. After this ELISA study, DNA inserts of clones that showed multiplereactivity with Toxoplasma gondii-positive sera and presented noreactivity with the negative sera were cloned as fusion products withthe protein Glutathione Sulphur Transferase (GST) and expressed in thecytoplasma of bacterial cells, for the purposes of determining theirspecificity and selectivity. To produce the fusion proteins each clonewas amplified from a single phage plaque by PCR, using the followingoligonucleotides: K47 5′-GGGCACTCGACCGGAATTATCG-3′ (SEQ ID No 37) andK85 5′-GGGTAAAGGTTTCTTTGCTCG-3′ (SEQ ID No 38). The resulting fragmentwas then purified by means of the “Qiagen Purification Kit” (Qiagen,Calif., USA), digested with the restriction enzymes SpeI and NotI andcloned in the vector pGEX-SN to generate the fusion with GST. Thecorresponding recombinant proteins were then expressed in E. coli andpurified by affinity using Glutathione-Sepharose resin (AmershamPharmacia Biotech, Sweden) and following standard protocols (Sambrook etal., 1989, Molecular Cloning, Cold Spring Harbor Laboratory Press, ColdSpring Harbor).

The following table 3, by way of examples, presents the reactivity withnegative and positive sera of a number of the clones selected, assayedin the form of fusion proteins:

TABLE 3 Reactivity of GST Reactivity of GST fusion protein fusionprotein with positive sera with negative sera Name of clone (pos./totalneg.) (neg./total neg.) Tx-4.11 32/40 1/28 Tx-15.11 22/40 0/28 Tx-1.1127/40 0/28 Tx-8.0 13/40 0/28 Tx-1.16 31/40 0/28 Tx-9.18  3/40 1/28Tx-7.11 39/40 3/28 Tx-4.18 21/40 1/28IgG Avidity for Determining the Time of Infection

Measurement of the binding force between immunoglobulin G (IgG) and theT. gondii-specific antigens (IgG avidity) is a diagnostic method used toestablish the time of infection: IgG avidity is lower during the acutephase of the infection and then tends to increase in the course of time(Hedman et al. 1989 J. Infect. Dis. 159, 736–740). Evaluation of IgGavidity is based on an enzyme-linked immunosorbent assay (ELISA) inwhich the immunoglobulins are “detached” from the antigen by washingwith a urea denaturing solution. A mathematical calculation based on thereactivity before and after the denaturing washing makes it possible toestimate serum IgG avidity (Jenum et al., 1997. J. Clin. Microbiol. 35,1972–1977). To evaluate the antigenic properties of the proteinfragments described in the present invention, an IgG avidity test basedon recombinant antigen fragments was developed and the results obtainedwere compared with the assay performed with a commercial kit (Toxo-IgGavidity kit, bioMerièux, France) that employs the whole parasite extractas antigen.

For the avidity analysis 27 sera coming from women who contractedprimary Toxoplasmosis during pregnancy and collected at different timesafter infection were used. The infection was diagnosed by seroconversionduring gestation, taking into consideration last negative and firstpositive samples. For each sample the specific IgG and IgM levels for T.gondii and the IgG avidity were determined by means of the use ofcommercial kits (LDBIO Diagnostic, France; bioMerièux, France).Multi-well plates (Nunc, Denmark) were incubated overnight at 4° C. witha solution of NaHCO₃ 50 mM, pH 9.6 containing the antigen fragmentsexpressed as GST fusion proteins at a final concentration of 5 μg/ml.The plates were blocked with 200 μl of blocking solution (5% skimmedmilk powder in PBS, 0.05% Tween-20) and then washed 5 times with washingbuffer (PBS, 0.05% tween-20). Serial dilutions of serum (1:50, 1:200,1:800, 1:3200) in 100 μl of blocking solution were incubated on platesfor 60 minutes at 37° C. The plates were then incubated with adenaturing solution of urea 6M in PBS/0.02% Tween-20 for 30 minutes at37° C. In parallel, for every sample, the same dilutions of serum wereeffected and the wells concerned were incubated with normal washingbuffer. 100 μl of blocking solution containing human anti-IgG antibodiesconjugated with the enzyme alkaline phosphatase (diluted 1:10000) wereadded to the plates. After 30 minutes' incubation at 37° C. the plateswere washed and the enzyme activity was determined with 100 μl ofdevelopment solution (10% diethanolamine pH 9.8, 0.5 mM MgCl₂, 0.05%NaN₃) containing the reaction substrate p-nitrophenylphosphate(Sigma-Aldrich, USA). The enzyme activity was measured at opticaldensities of 405 nm and 620 nm by means of an automatic ELISA OD reader(Multiskan Labsystem, Finland) and the avidity calculation was doneaccording to the mathematical analysis described in the literature(Jenum et al., 1997. J. Clin. Microbiol. 35, 1972–1977).

The following table 4 gives, by way of examples, the avidity of thehuman sera for a number of the antigen fragments selected.

The values should be interpreted as follows (commercial kit criterion):

<15% Low avidity:acute infection in the last three months

15%–30% Borderline avidity:probable primary infection within 3–6 monthsispossible

>30% High avidity:excludes primary infection within the last threemonths

EXAMPLE 2

Using the vector λKM4 of Example 1, a library of DNA fragments of knownToxoplasma gondii genes was constructed.

Cells of Toxoplasma gondii (10⁶ parasites, strain ME49) were grown invitro in monkey kidney cells (“VERO” African green monkey cells) usingDMEM culture medium containing 10% foetal bovine serum, 2 mM glutamineand 0.05 mg/ml gentamicin (Gibco BRL, Canada). To have both forms of theparasite (tachyzoites and bradyzoites) present in the cell cultures, anexperimental protocol was used based on the change in pH of the culturemedium (Soete et al., 1994, Experimental Parasitology, 78, 361–370). Theparasites were collected after complete lysis of the host cells andpurified by filtration (filter porosity 3 μm) followed by centrifuging.2 μg of mRNA were isolated from 5×10⁶ parasites using the “QuickPrepMicro mRNA Purification Kit” (Amersham Pharmacia Biotech, Sweden) andfollowing the manufacturer's instructions. cDNA was synthesised from 200ng of poly(A)+ RNA using the “SMART cDNA Library Construction Kit”(Clontech, CA, USA) and following the manufacturer's instructions.Genomic DNA was purified from the remaining 5×10⁶ cells using standardprocedures (Sambrook et al., 1989, Molecular Cloning: a laboratorymanual, Cold Spring Harbor Laboratory Press, NY) and stored at −20° C.

For the construction of the expression/exposure library the followinggenes, expressed only in the bradyzoite stage, were amplified by meansof PCR with specific oligonucleotides:

-   1—SAG2D (Lekutis et al., 2000, Experimental Parasitology, 96, 89–96)    was obtained from genomic DNA using the oligonucleotides    5′-ATGGCGGCTGCACACTCG-3′ (SEQ ID No 39) and    5′-GAACATATTCCCTGTCACCAATG-3′ (SEQ ID No 40);-   2—SAG4 (Odberg-Ferragut et al., 1996, Molecular and Biochemical    Parasitology, 82, 237–244) was obtained from genomic DNA using the    oligonucleotides 5′-ATGACGAAAAATAAAATTCTTCTC-3′ (SEQ ID No 41) and    5′-CATTGATATCAACACAAAGGCC-3′ (SEQ ID No 42)-   3—BSR4 (Manger et al., 1998, Infection and Immunity, 66, 2237–2244)    was obtained from genomic DNA using the oligonucleotides    5′-ATGGTGATGATGGGCAGCATG-3′ (SEQ ID No 43) and    5′-CGGCGGCCGCGCTAGAGG-3′ (SEQ ID No 44);-   4—MAG1 (Parmley et al., 1994, Molecular and Biochemical    Parasitology, 66, 283–296) was obtained from genomic DNA using the    oligonucleotides 5′-CGTTGGATCCTTGGATTGAGCCAAAGGGTGCCAG-3′ (SEQ ID    No 45) and 5′-CCCAGAATTCTCAAGCTGCCTGTTCCGCTAAGATCTG-3′ (SEQ ID No    46);-   5—LDH2 (Yang and Parmley, 1997, Gene, 184, 1–12) was obtained from    cDNA using the oligonucleotides 5′-ATGACGGGTACCGTTAGCA-G-3′ (SEQ ID    No 47) and 5′-ACCCAGCGCCGCTAAACTC-3′ (SEQ ID No 48);-   6—ENO1 (Dzierszinski et al., 2001, Journal of Molecular Biology,    309, 1017–1027) was obtained from genomic DNA using the    oligonucleotides 5′-ATGGTGGTTATCAAGGACATCG-3′ (SEQ ID No 49) and    5′-TTTTGGGTGTCGAAAGCTCTC-3′ (SEQ ID No 50);-   7—BAG1 (Bohne et al., 1995, Molecular Microbiology, 16, 1221–1230)    was obtained from cDNA using the oligonucleotides    5′-ATGGCGCCGTCAGCATCG-3′ (SEQ ID No 51) and    5′-CTTCACGCTGATTTGTTGCTTTG-3′ (SEQ ID No 52);-   8—p-ATPase (Holpert et al., 2001, Molecular and Biochemical    Parasitology, 112, 293–296) was obtained from genomic DNA using the    oligonucleotides 5′-ATGGACGAAGCGAGCAGAAGG-3′ (SEQ ID No 53) and    5′-ACGCGTGATCGAAGGAACCG-3′ (SEQ ID No 54).

10 μg of DNA deriving from a mixture of the amplification products ofthe above-mentioned genes were fragmented randomly using 0.5 ng of theendonuclease DNaseI (Sigma-Aldrich, USA). The mixture of DNA and DNaseIwas incubated for 20 minutes at 15° C. and the DNA fragments werepurified by means of the “QIAquick PCR Purification Kit” (Qiagen,Calif., USA), following the manufacturer's instructions. The 3 μg endsof the cDNA fragments were “flattened” by incubating the DNA with 9units of the enzyme T4 DNA polymerase (New England Biolabs, MA, USA) for60 minutes at 15° C. The fragments were then purified by means ofextraction in phenol/chloroform and subsequent precipitation in ethanol.500 ng of the resulting DNA were bound with a 20-fold molar excess of“synthetic adaptors” for the purposes of adding the restriction sitesSpeI and NotI to the ends of the fragments. Six adaptors were used,obtained by hybridisation of the following pairs of oligonucleotides:K185 5′-CTAGTCGTGCTGGCCAGC-3′ (SEQ ID No 5) and K1865′-GCTGGCCAGCACGA-3′ (SEQ ID No 6); K187 5′-CTAGTCGTGCTGGCCA GCT-3′ (SEQID No 7) and K188 5′-AGCTGGCCAGCACGA-3′ (SEQ ID No 8); K189 5′-CTAGTCGTGCTGGCCAGCTG-3′(SEQ ID No 9) and K190 5′-CAGCTGGCCAGCACGA-3′ (SEQ ID No10); K191 5′-TCTGGTGGCGGTAGC-3′ (SEQ ID No 11) and K1925′-GGCCGCTACCGCCACCAGA-3′(SEQ ID No 12); K193 5′-TTCTGGTGGCGGTAGC-3′(SEQ ID No 13) and K194 5′-GGCCGCTACCGCCACCAGAA-3′ (SEQ ID No 14); K1955′-TTTCTGGTGGCGGTAGC-3′ SEQ ID No 15) and K1965′-GGCCGCTACCGCCACCAGAAA-3′ (SEQ ID No 16). The excess of unligatedadaptors was removed from the ligation mixture by electropheresis on 2%agarose gel and the cDNA fragments with molecular weights ranging from250 bp to 1000 bp were excised from the gel and purified by means of the“Qiaquick gel extraction kit” (Qiagen, Calif., USA) following themanufacturer's instructions. The vector λKM4 was digested with SpeI/NotIand for the construction of the library 6 ligation mixtures wereperformed, each containing 0.4 μg of vector and approximately 7 ng ofinsert. After overnight incubation at 4° C. the ligation mixtures werepackaged in vitro with the “Ready-To-Go lambda packaging kit” (AmershamPharmacia Biotech, Sweden) and plated for infection of BB4 cells(bacterial cells of E. coli strain BB4; Sambrook et al., 1989, MolecularCloning: a laboratory manual, Cold Spring Harbor Laboratory Press, NY).After overnight incubation at 37° C. the phage was eluted from theplates with SM buffer (Sambrook et al., 1989, Molecular Cloning: alaboratory manual, Cold Spring Harbor Laboratory Press, NY), purified,concentrated and stored at −80° C. in SM buffer containing 7%dimethylsulphoxide. The complexity of the library calculated as thenumber of total independent clones with inserts was 10⁶ clones.

Affinity selection, phage-ELISA, immunoscreening and phage clonespreparation were performed exactly as described in Example 1.

The following table 5 gives, by way of examples, the reactivity of anumber of the recombinant bacteriophages selected.

TABLE 5 Reactivity of phage Reactivity of phage clone with positiveclone with negative sera (positive/ sera (negative/ Name of clone totalpositive) total negative) TxB-cl21.2 10/20 1/10 TxB-cl26.3 12/20 0/10TxB-44.3 10/20 0/10 TxB-7.1  8/20 0/10 TxB-9.1  1/20 1/10 TxB-12.1  5/200/10Characterisation of Positive Clones

The clones which showed multiple reactivity with the Toxoplasma gondiipositive sera and which presented no reactivity with the negative serawere subsequently sequenced and compared with the sequences of the genesused to construct the library.

The following table 6 gives, by way of examples, the sequences of someof the clones selected:

TABLE 6 Name of clone Sequence TxB-26.3GGATTGAGCCAAAGGGTGCCAGAGCTACCAGAAGT (SEQ ID No 55)GGAGCCCTTTGATGAAGTAGGCACGGGAGCTCGAC GGTCCGGGTCCATTGCGACCCTTCTTCCACAAGACGCTGTTTTATATGAGAACTCAGAGGACGTTGCCGT TCCGAGTGATTCAGCATCGACCCCGTCATACTTTCATGTGGAATCTCCAAGTGCTAGTGTGGAAGCCGCG ACTGCCGCTGTGGGAGAGGTGGTGCCGGACTGTGAAGAACAACAGGAACAGGGTGACACGACGTTATCCG ATCACGATTTCCATTCA TxB-cl7.1TCTTCAGAAAGATGACGTAACCATAGAAGTCGACA (SEQ ID No 56)ACGGAGCCATCGTTATCAAAGGAGAGAAGACCTCG AAAGAAGCGGAGAAAGTGGACGATGGCAAAACAAAGAACATTTTGACTGAGCGAGTGTCCGGTTATTTTG CGCGCCGGTTCCAGCTCCCGAGTAATTACAAGCCCGACGGAATCAGTGCGGCAATGGACAACGGCGTTCT ACGTGTCACGATCAAGGTCGAGGATTCAGGGGGCGCAAAGCAACAAATCAGCGTG

The clone TxB-cl26.3 constitutes a fragment of the gene MAG1, a 65 kDaprotein of the matrix and wall of T. gondii cysts (Parmley et al., 1994,Molecular and Biochemical Parasitology, 66, 283–296), the proteinproduct of which has never been identified as an “antigen fragment” inthe human humoral response. Said clone has the amino acid sequenceGLSQRVPELPEVEPFDEVGTGARRSGSIATLLPQDAVLYENSEDVAVPSDSASTPSYFHVESPSASVEAATGAVGEVVPDCEEQQEQGDTTLSDHDFH (SEQ ID No 57) and its use as a fragment containing anepitope is covered by the present invention.

The clone TxB-cl7.1 constitutes a fragment of the gene BAG1, a 30 kDaprotein of the heat shock protein family of T. gondii (Bohne et al.,1995, Molecular Microbiology, 16, 1221–1230), the protein product ofwhich has never been identified as an “antigen fragment” in the humanhumoral response. Said clone has the amino acid sequenceLNPIDDMLFETALTANEMMEDITWRPRVDVEFDSKKKEMIILADLPGLQKDDVTIEVDNGAIVIKGEKTSKEAEKVDDGKTKNILTERVSGYFARRFQLPSNYKPDGISAAMDNGVLRVTIKVEDSGGAKQQISV (SEQ ID No 58) and its useas a fragment containing an epitope is covered by the present invention.

Expression of DNA Fragments Selected from the Library as Fusion Productswith GST

The phage clones for which specific reactivity with sera of patientstesting positive for Toxoplasma gondii was demonstrated, were amplifiedand then analysed with a substantial panel of positive and negativesera. After this ELISA study, the clones that showed multiple reactivitywith Toxoplasma gondii-positive sera and presented no reactivity withthe negative sera were cloned as fusion products with the proteinGlutathione Sulphur Transferase (GST) and expressed in bacterial cells,for the purposes of determining their specificity and selectivity. Toproduce the fusion proteins each clone was amplified from a single phageplaque by PCR, using the following oligonucleotides: K475′-GGGCACTCGACCGGAATTATCG-3′ (SEQ ID No 37) and K855′-GGGTAAAGGTTTCTTTGCTCG-3′ (SEQ ID No 38). The resulting fragment wasthen purified by means of the “Qiagen Purification Kit” (Qiagen, Calif.,USA), digested with the restriction enzymes SpeI and NotI and cloned inthe vector pGEX-SN to generate the fusion with GST. The correspondingrecombinant proteins were then expressed in E. coli and purified byaffinity using Glutathione-Sepharose resin (Amersham Pharmacia Biotech,Sweden) and following standard protocols (Sambrook et al., 1989,Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold SpringHarbor).

The following table 7, by way of examples, presents the reactivity withnegative and positive sera of a number of the clones selected, assayedin the form of fusion proteins:

TABLE 7 Reactivity of GST Reactivity of GST fusion protein with fusionportein with positive sera negative sera Name of clone (pos./total neg.)(neg./totale neg.) TxB-cl26.3 30/34 0/32 TxB-cl7.1 17/34 0/32

EXAMPLE 3

By using the same strategy described in Example 2, a gene collection ofDNA encoding for protein products of the Toxoplasma gondii micronemefamily was used to construct a “microneme-display library”.

For the construction of the microneme-library the following genes wereamplified by means of PCR with specific oligonucleotides:

-   1—MIC2 (Wan et al, 1997, Mol. Biochem. Parasitol. 84: 203–214) was    obtained from single strand cDNA using the oligonucleotides    5′-ATGAGACTCCAACCGAGGCC-3′ (SEQ ID No 69) and    5′-CTGCCTGACTCTTTCTTGGACTG-3′ (SEQ ID No 70);-   2—M2AP (Rabenau et al., 2001, Mol. Microbiol. 41: 537–547) was    obtained from single strand cDNA using the oligonucleotides    5′-GGAAAGTTGGAAATCCGGCGGC-3′ (SEQ ID No 71) and    5′-CGCCTCATCGTCACTCGGC-3′ (SEQ ID No 72)-   3—MIC4 (Brecht et al., 2001, J. Biol. Chem. 276:4119–412) was    obtained from single strand cDNA using the oligonucleotides    5′-ATGAGAGCGTCGCTCCCGG-3′ (SEQ ID No 73) and    5′-GTGTCTTTCGCTTCAAGCACCTG-3′ (SEQ ID No 74);-   4—AMA1 (Hehl et al., 2000, Infect. Immun. 68:7078–7086) was obtained    from single strand cDNA using the oligonucleotides    5′-ATGGGGCTCGTGGGCGTAC-3′ (SEQ ID No 75) and    5′-GATCAACGCAGTGTTAGAGCCAC-3′ (SEQ ID No 76);

10 μg of DNA deriving from a mixture of the amplification products ofthe above-mentioned genes were fragmented randomly using 0.5 ng of theendonuclease DNaseI (Sigma-Aldrich, USA). The mixture of DNA and DNaseIwas incubated for 20 minutes at 15° C. and the DNA fragments werepurified by means of the “QIAquick PCR Purification Kit” (Qiagen,Calif., USA), following the manufacturer's instructions. Consequentsteps for the construction of the microneme-library, and for theaffinity selection were performed by following the procedure describedin Example 2.

Selection of the Microneme-Library with Sera of Infants Who WereInfected by T. gondii During Pregnancy

To identify the antigenic domains of the T. gondii microneme proteins anaffinity selection procedure was used consisting of two “panning” cycleswith four sera collected from infants who were congenitally infected bythe parasite, followed by an immunological screening procedure carriedout with the same sera. The library was selected with sera T1, T2, T3,T4, generating, after a single selection cycle, the correspondingmixtures p1^(I), p2^(I), p3^(I) and p4^(I). Each mixture was thensubjected to a second affinity selection cycle with the same serum,giving rise to a second series of mixtures (called p1^(II)p2^(II),p3^(II) and p4^(II)). Various positive clones were identified by meansof immunoplate screening per plaque of reactive mixtures.

Phage-Elisa, immunoscreening, and the preparation of phage clones weresubsequently performed exactly as described in Examples 1 and 2.

The following table 8 gives, by way of examples, the reactivity of anumber of the recombinant bacteriophages selected.

TABLE 8 Reactivity of phage Reactivity of phage clone with positiveclone with negative sera (positive/ sera (negative/ Name of clone totalpositive) total negative) Tx-2.a 13/16 0/10 Tx-1.b 11/16 0/10 Tx-11.b12/16 0/10 Tx-13.b  9/16 0/10 Tx-15.b  9/16 0/10Characterisation of Positive Clones

The following table 9 gives the sequences of the clones selected:

TABLE 9 Name of the Identi- Classi- clone Sequence fication ficationTx-2.a CCCCAGGATGCCATTTGCTCGGATT MIC2 Micro- (SEQ IDGGTCCGCATGGAGCCCCTGCAGTGT neme No 59) ATCCTGCGGTGACGGAAGCCAAATC proteinAGGACGCGAACTGAGGTTTCTGCTC unknown CGCAACCTGGAACACCAACATGTCC as anti-GGACTGCCCTGCGCCCATGGGAAGG gen in ACTTGCGTGGAACAAGGCGGACTTG humanAAGAAATCCGTGAATGCAGTGCGGG response GGTATGTGCTGTTGACGCTGGATGTGGCGTCTGGGTT Tx-1.b CCGTGTCCAATTAATGCAACTTGCG MIC2 Micro- (SEQ IDGTCAGTTTGAAGAATGGAGTACATG neme No 60) CTCGGTCTCATGTGGTGGTGGACTG proteinAAAACGAGGTCGAGGAACCCTTGGA unknown ATGAAGACCAACAACATGGAGGACT as anti-ATCCTGCGAGCAGCAGCATCCTGGT gen in GGGCGGACGGAAACGGTAACTTGCA humanATCCTCAAGCGTGTCCTGTGGATGA response ACGACCGGGGGAGTGGGCAGAGTGGGGGGAATGTAGTGTCACGTGCGGCG ACGGAGTGCGAGAGCGCAGGCGCGGGAAAAGTCTAGTTGAGGCTAAATTC GGCGGACGCACCATTGATCAGCAGAATGAGGCTCTTCCGGAAGACTTAAA AATCAAAAACGTCGAGTATGAGCCATGTTCGTATCCTGCTTGTGGAGCTT CCTGCACGTACGTCTGGAGTGACTG GAACAAG Tx-11.bAACGAACCGGTGGCCCTAGCTCAGC M2AP Micro- (SEQ ID TCAGCACATTCCTCGAGCTCGTCGAneme No 61) GGTGCCATGTAACTCTGTTCATGTT protein CAGGGGGTGATGACCCCGAATCAAAunknown TGGTCAAAGTGACTGGTGCAGGATG as anti- GGATAATGGCGTTCTCGAGTTCTAT genin GTCACGAGGCCAACGAAGACAGGCG human GGGACACAAGCCGAAGCCATCTTGC responseGTCGATCATGTGTTATTCCAAGGAC ATTGACGGCGTGCCGTCAGACAAAGCGGGAAAGTGCTTTCTGAAGAACTT TTCTGGTGAAGACTCGTCGGAAATAGACGAAAAAGAAGTATCTCTACCCA TCAAGAGCCACAACGATGCGTTCATGTTCGTTTGTTCTTCAAATGATGGA TCCGCACTCCAGTGTGATGTTTTCGCCCTTGATAACACCAACTCTAGCGA CGGGTGGAAAGTGAATACCGTGGATCTTGGCGTCAGCGTTAGTCCGGATT TGGCATTCGGACTCACTGCAGATGGGGTCAAGGTGAAGAAGTTGTACGCA AGCAGCGGCCTGACAGCGATCAACGACGACCCTTCCTTGGGGTGCAAGGC TCCTCCCCATTCTCCGCCGGCCGGAGAGGAACCGAGTTTGCCGTCGCCTG AAAACAGCGGGTCTGCAACACCAGCGGAAGAAAGTCCGTCTGAGTCTGAA TCT Tx-13.b CTTCGCGGGTACAGGTTCGGTGTTT AMA1Micro- (SEQ ID GGAAGAAAGGCCGTTGCCTCGACTA neme No 62)CACTGAATTGACCGACACTGTGATA protein GAACGTGTTGAGTCAAAGGCACAGT unknownGCTGGGTGAAAACCTTTGAAAACGA as anti- CGGGGTCGCGAGTGACCAACCCCAT gen inACGTATCCACTGACGTCGCAAGCAT human CATGGAACGATTGGTGGCCTCTCCA responseCCAGAGTGACCAACCTCACTCAGGT GGCGTTGGGCGTAATTACGGTTTCTACTACGTGGACACGACTGGAGAGGG CAAGTGTGCACTCTCTGACCAGGTACCCGACTGCCTGGTGTCGGATTCTG CCGCCGTGTCGTATACAGCAGCGGGGAGTTTGTCTGAAGAGACGCCGAAT TTCATAATTCCGTCAAATCCCTCTGTTACTCCGCCAACGCCCGAGACGGC ACTTCAGTGCACGGCCGACAAGTTCCCCGACTCTTTCGGTGCCTGCGACG TTCAAGCGTGTAAAAGACAGAAGACGTCCTGCGTTGGCGGACAGATTCAA AGTACTAGCGTCGACTGCACCGCGGACGAACAAAATGAATGTGGCTCTAA CACTGCG Tx15.b AGTGCCAACGTAACAAGTTCGGAGC MIC4Micro- (SEQ ID CTGCAAAACTTGATCTCTCTTGTGC neme No 63)GCACTCTGACAATAAGGGATCAAGG protein— GCTCCCACAATAGGCGAGCCAGTGC unknownCAGATGTGTCCCTGGAACAATGTGC as anti- TGCGCAATGCAAGGCTGTTGATGGC gen inTGCACACATTTCACTTATAATGACG human ATTCGAAGATGTGCCATGTGAAGGA responseGGGAAAACCCGATTTATACGATCTC ACAGGAGGCAAAACAGCACCGCGCAGTTGCGATAGATCATGCTTCGAACA ACACGTATCGTATGAGGGAGCTCCTGACGTGATGACAGCGATGGTCACGA GCCAGTCAGCGGACTGTCAGGCTGCGTGTGCGGCTGACCCGAGCTGCGAG ATCTTCACTTATAACGAACACGACCAGAAATGTACTTTCAAAGGAAGGGG GTTTTCTGCGTTTAAGGAACGAGGGGTGTTGGGTGTGACTTCCGGGCCGA AACAGTTCTGCGATGAAGGCGGTAA ATTAACT

The clones Tx-2.a e Tx-1.b represent two distinct fragments of the MIC2gene (Wan et al, 1997, Mol. Biochem. Parasitol. 84: 203–214) and havenever been identified as antigens of the human antibody response. Saidclones have respectively the amino acid sequencesPQDAICSDWSAWSPCSVSCGDGSQIRTRTEVSAPQPGTPTCPDCPAPMGRTCVEQGGLEEIRECSAGVCAVDAGCGVWV (SEQ ID No 64) andPCPINATCGQFEEWSTCSVSCGGGLKTRSRNPWNEDQQHGGLSCEQQHPGGRTETVTCNPQACPVDERPGEWAEWGECSVTCGDGVRERRRGKSLVEAKFGGRTIDQQNEALPEDLKIKNVEYEPCSYPACGASC TYVWSDWNK (SEQ ID No 65)and their use as fragments containing an epitope is covered by thepresent invention.

The clone Tx-11.b represents a distinct fragment of the M2AP gene(Rabenau et al., 2001, Mol. Microbiol. 41: 537–547) and has never beenidentified as antigen of the human antibody response. Said clone has theamino acid sequenceNEPVALAQLSTFLELVEVPCNSVHVQGVMTPNQMVKVTGAGWDNGVLEFYVTRPTKTGGDTSRSHLASIMCYSKDIDGVPSDKAGKCFLKNFSGEDSSEIDEKEVSLPIKSHNDAFMFVCSSNDGSALQCDVFALDNTNSSDGWKVNTVDLGVSVSPDLAFGLTA DGVKVYASSGLTAINDDPSLGCKAPPHSPPAGEEPSLPSPENS GSATPAEESPSESES (SEQ ID No 66)and its use as fragment containing an epitope is covered by the presentinvention.

The clone Tx-13.b represents a fragment of the AMA1 gene (Hehl et al.,2000, Infect. Immun. 68:7078–7086). Said clone has the amino acidsequenceLRGYRFGVWKKGRCLDYTELTDTVIERVESKAQCWVKTFENDGVASDQPHTYPLTSQASWNDWWPLHQSDQPHSGGVGRNYGFYYVDTTGEGKCALSDQVPDCLVSDSAAVSYTAAGSLSEETPNFIIPSNPSVTPPTPETALQCTADKFPDSFGACDVQACKRQKTSCVGGQIQ STSVDCTADEQNECGSNTA (SEQID No 67) and its use as fragment containing an epitope is covered bythe present invention.

The clone Tx-15.b represents a fragment of the MIC4 gene (Brecht et al.,2001, J. Biol. Chem. 276:4119–4127). Said clone has the amino acidsequenceSANVTSSEPAKLDLSCAHSDNKGSRAPTIGEPVPDVSLEQCAAQCKAVDGCTHFTYNDDSKMCHVKEGKPDLYDLTGGKTAPRSCDRSCFEQHVSYEGAPDVMTAMVTSQSADCQAACAADPSCEIFTYNEHDQKCTFKGRGFSAFKERGVLGVTSGPKQFCDEGGKLT (SEQ ID No 68) and its use asfragment containing an epitope is covered by the present invention.

Expression of DNA Fragments Selected from the Microneme-Library asFusion Products with GST

Phage clones isolated from the microneme-library were cloned as fusionproducts with GST protein and expressed in bacterial cells, for thepurposes of determining their specificity and selectivity. The proceduredescribed in Examples 1 and 2 was used to produce the fusion proteins.

The following table 10, by way of example, presents the reactivity withnegative and positive sera of a number of the clones selected, assayedin the form of fusion proteins:

TABLE 10 Reactivity of GST Reactivity of GST fusion protein fusionportein with positive sera with negative sera Name of clone (pos./totalneg.) (neg./totale neg.) Tx-2.a 29/30 0/15 Tx-1.b 15/30 0/15 Tx-11.b23/30 0/15 Tx-13.b 24/30 0/15 Tx-15.b 12/30 0/15

1. An isolated polypeptide consisting of an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 31, 58, 26, 28, 29, 64, 65, 67,66, 57, 34, 33, 32, 68, 30 and
 27. 2. A pharmaceutical compositioncomprising a polypeptide of claim
 1. 3. An isolated polypeptideconsisting of the amino acid sequence of SEQ ID NO:26.
 4. A compositioncomprising the polypeptide of claim 3.